Drug-resistance is rapidly spreading among Gram-negative bacteria, seriously complicating the treatment of infected patients and increasing the risk of lethal outbreaks. In particular, carbapenem-resistant Enterobacteriaceae (CRE), organisms associated with high mortality rate (>40%), have become prevalent worldwide. During 2012, 18% of long-term acute care hospitals in the U.S had at least one case of CRE. Accurate and rapid identification of drug-resistance is fundamental for proper treatment and prevention of nosocomial spread. However, microbiology laboratories have deficient tools for resistance diagnosis. Phenotypic assays are the most common diagnostic methods, but they are slow (24 hours), frequently inconclusive and may fail to detect carbapenemases. Alternative methods based on nucleic acid detection exist, however, these methods are either time consuming and require trained personnel, or only cover a limited number of resistance determinants. Here, we will develop a novel class of microarrays based on Twist-Sensor technology for rapid, accurate and multiplex detection of drug resistance. Twist-Sensor is a novel technology for nucleic acid detection based on single molecule DNA hybridization and supercoiling. In this technique, the hybridized targets are subjected to disrupting torsional stres which allows for rapid detection of multiple target sequences with extremely low background noise using a simple device. We will demonstrate the capabilities of Twist-Sensor technology by developing a microarray able to detect multiple carbapenemase genes in a procedure with minimum hands- on time and with a total processing time of less than 2 hours. Aim 1 will demonstrate that Twist- Sensor technology is capable of detecting a resistance determinant in bacterial cultures without DNA amplification. Aim 2 will focus on developing a Twist-Sensor detection mechanism that is easy to automate, inexpensive and has high throughput. We will use this novel microarray to develop a multiplex assay for detection of the most prevalent carbapenemases (KPC, NDM, OXA-48, VIM and IMP). In order to optimize and characterize the functionality of the new microarray, we will analyze bacterial culture samples of resistant isolates.